It is not practical to conduct surgery while viewing only the fluorescence of a contrast agent. Instead, fluorescence images need to be superimposed continuously in real-time and in precise spatial register with reflectance images showing the morphology of the normal tissue and the location of the surgical instruments. Reflectance is inherently brighter than fluorescence, in that the fraction of incident photons returned by reflectance is much higher than that from fluorescence (even from strongly labeled tissue). Also, fluorescence requires spectral separation between bright short-wavelength excitation and relatively dim longer-wavelength emission, filtered to remove reflected excitation wavelengths.
The simplest approach to overlaying a reflectance image with a fluorescent one does not require a camera at all—the image is simply viewed by the eye. This approach is feasible primarily for dyes excited in the violet or blue, with excitation and emission filters tailored to provide a mixture of reflectance and fluorescence directly visible by the surgeon. An early example was the visualization of the fluorescence from protoporphyrin IX (Leica FL400-excitation at 380-420 nm, display at 480-720 nm and Zeiss OPMI Pentero—excitation 400-410 nm, display 620-710 nm) generated in tumors by systemic administration of the metabolic precursor, 5-aminolevulinic acid (Stummer, W. et al. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. The lancet oncology 7: 392-401 (2006)). In both these examples, the red emission filter was deliberately designed to leak at shorter wavelengths so that normal tissue landmarks and surgical instruments were visible by the violet reflectance light while the cancer tissue glowed red. Normal full color reflectance was sacrificed so that image processing could be completely avoided. Similarly, fluorescein fluorescence (Zeiss Pentero—excitation 488 nm, emission >500 nm) was viewed with an excitation filter mainly passing blue but with some leakage of green and red, paired with an emission filter preferentially passing green but with slight leakage of blue and somewhat more of red. The leakages allow blue, green, and red reflectances to generate a reasonable white-light reflectance image with green fluorescence superimposed. The advantage of these approaches is that the only modification of standard equipment is the inclusion of judiciously tailored excitation and emission filters. The disadvantages are 1) inflexibility, in that the filters have to be optimized for a given tissue concentration of a given dye, 2) spectral distortion of the reflectance image (in the worst case, confinement to violet), and 3) complete reliance on the surgeon's subjective color discrimination to decide how much fluorescence is sufficient to warrant resection.
The next level of sophistication for overlaying a reflectance image with a fluorescent one requires the use of two cameras and therefore cannot be viewed directly by eye. With this approach, two cameras collect two separate images, one for the white light reflectance image and one for the fluorescence image. Separate cameras mean that the gain for each image can be controlled independently and that the reflectance camera no longer has to look through the emission filter of the fluorescence camera. This is the obvious approach (De Grand, A. M. & Frangioni, J. V. An operational near-infrared fluorescence imaging system prototype for large animal surgery. Technology in cancer research & treatment 2: 553-62 (2003) and Troyan, S. L. et al. The FLARE intraoperative near-infrared fluorescence imaging system: a first-in-human clinical trial in breast cancer sentinel lymph node mapping. Annals of surgical oncology 16: 2943-52 (2009)) when the fluorophore excitation and emission wavelengths are in the NIR, because the surgical field can be illuminated with white visible light (400-650 nm) plus 760 nm in the case of indocyanine green. The reflectance camera sees the white light reflectance but not the NIR excitation, while the fluorescence camera sees the 800 nm emission through a suitable long-pass filter (Troyan, S. L. et al. The FLARE intraoperative near-infrared fluorescence imaging system: a first-in-human clinical trial in breast cancer sentinel lymph node mapping. Annals of surgical oncology 16: 2943-52 (2009)). A bit more ingenuity is required when the fluorophore excitation and emission are at visible wavelengths. One solution is to confine the excitation to three narrow spectral lines of blue, green, and red, say 488, 543, and 633 nm (Themelis, G., Yoo, J. S. & Ntziachristos, V. Multispectral imaging using multiple-bandpass filters. Optics letters 33: 1023-5 (2008)) whose relative intensities are adjusted to generate a reasonable simulation of white light for the reflectance camera. Ideally one of these wavelengths should be optimal for exciting the fluorophore. Meanwhile the fluorescence camera looks through an emission filter selective for one or more of the wavelength gaps between the sharp excitation lines. Depending on the fluorophore, some sacrifice of emission bandpass is likely to be necessary to avoid interference from the next longer illumination line.
Fluorescence Guided Surgery is a method of enhancing visual contrast for specific tissues and organs during surgery, providing an important tool for extending the visual differentiation between tissue targeted for excision and tissue intended for preservation beyond that which is available with white light alone. Examples of such relevant targets include tumors, nerves, and blood vessels (Orosco, R., Tsien, R. & Nguyen, Q. Fluorescence Imaging in Surgery. IEEE Reviews in Biomedical Engineering 6: 178-187 (2013) and Nguyen, Q. & Tsien, R. Fluorescence Guided Surgery with Live Molecular Navigation—A New Cutting Edge (Strategies to improve surgery). Nature Reviews Cancer 13: 653-62 (2013)).
This present disclosure and invention addresses the problem of simultaneously viewing of 1) the surgical field with white light reflectance (what all surgeons are trained to do) in conjunction with 2) fluorescently labeled targets. The present disclosure provides methods for achieving optical contrast between the fluorescently labeled target and the remainder of surgical field seen with white light reflectance by overlaying a fluorescent image of the surgical field with a live color image of the same field. The fluorescent portion of the image is pseudocolored with a hue that is not normally present in mammalian tissue; allowing the surgeon to easily distinguish between normal tissue and targeted tissue. This invention is designed to work with any fluorophore, at any excitation or emission wavelength. The invention can handle multiple individual fluorophores as well as fluorescence resonance energy transfer (FRET) fluorophore pairs, simultaneously with white light reflectance. In addition the, invention can be adapted to systems with binocular vision for improved depth of field during surgery.